(a) Western blot of splenic membrane and serum proteins. (b) Western blot analysis of the IgD glycosylation in bovine serum. 1, Untreated serum proteins; 2, endo-α-N-acetylgalactosaminidase-treated serum proteins; 3, PNGase-treated serum proteins. The primary antibody 13C2 was diluted at 1∶200 or 1∶400. (c) Serum immunoblot analysis with mAb 13C2 and anti-bovine IgM polyclonal antibody under nonreducing conditions. The arrow indicates the monomer of IgD. (d) Immunoblot detection of serum IgD and IgM in differently aged cows. 1, 60 days old; 2, 180 days; 3, 1 year; 4, 2 years; 5, 3 years; 6, 4 years; 7, 5 years; 8, 6 years. The arrows indicate the IgD heavy chain.